Glycosaminoglycans (GAGs) are complex linear polysaccharides of the extracellular matrix (ECM). GAG's are characterized by repeating disaccharide structures of an N-substituted hexosamine and a uronic acid, {hyaluronan(HA), chondroitin sulfate (CS), chondroitin (C), dermatan sulfate (DS), heparan sulfate (HS), heparin (H)], or a galactose, keratan sulfate (KS)}. Except for HA, all exist covalently bound to core proteins. The GAGs with their core proteins are structurally referred to as proteoglycans (PGs).
Chondroitin and dermatan sulfate are by far the most common glycosaminoglycans of the vertebrate body. Classification of chondroitin sulfates are: 1) CS-A (chondroitin-4-sulfate), 2) CS-B, or DS, containing 4-sulfated N-acetyl-galactosamine and iduronic acid, and 3) CS-C (chondroitin-6-sulfate). Whereas the amino acid sequences, conformation, and biological functions of many of the CS-PG core proteins are now well established the precise structure of the GAG polymers are not firmly established. CS is comprised of between 15 and 50 disaccharide units of repeating beta-linked D-glucuronic acid and N-acetyl-D-galactosamine, the latter sulfated at the 4- or 6-position. Sulfation does not appear to be random, as assumed earlier. Repeating sulfation patterns have been detected, as well as other forms of repeating microheterogeneity including glucuronate-iduronate uronic acid epimerizations. The lack of reliable tools have to date, precluded the characterization. The bacterial chondroitinases exhibit cleavage specificity for glucuronate or iduronate residues (i.e. chondroitinase AC II and B, respectively). Unlike bacterial keratanases and heparinases, bacterial chondroitinases lack selective cleavage sites for specific sulfation sequences. Vertebrate chondroitinases may in fact have some form of such cleavage specificity and may be of critical importance for therapeutic intervention, as pharmaceuticals.
The CS- and DS-PGs are important ECM structures with both regulatory and structural roles. These molecules play both major structural components of bone, tendon, cartilage, scars, and fibrous connective tissue. CSPG's are also found in lesser amounts in most tissues, where they function as regulatory and signaling molecules involved in adhesion, migration, differentiation, and proliferation (Vogel, 1994). They bind growth factors, function as receptors, adhesion molecules, regulate deposition and distribution of other ECM polymers, and are themselves building blocks (Iozzo, 1998). Examples of these molecules include aggrecan, biglycan, brevican, decorin, neurocan, serglycin and versican. The peptide bikunin has a covalently bound CS chain, necessary for its activity as an inter-alpha-trypsin inhibitor (Yamada et al., 1995), and as a hyaluronidase inhibitor (Mio et al., 2000). CS-PGs also bind integrins, initiating cascades of signal transduction events (Iida et al., 1996; Li et al., 2000). A CS-PG is also a cell surface receptor for the malarial parasite in the human placenta (Valiyaveettil et al., 2001).
Following spinal cord injury, glial scars are produced by astrocytes and contain chondroitin sulfate proteoglycans (CSPGs). CSPGs play a crucial role in the inhibition of axon growth (Levine, 1994; Powell et al., 1997). For example, during fetal development, CSPGs repel axons and inhibit neural cell adhesion. CSPG's also play an important role in boundary formation (Snow et al., 1990, 1992; Powell and Geller, 1999). In addition the expression of CSPG increases following injury of CNS (McKeon et al., 1991; Davies et al., 1997).
Studies indicate that the inhibitory effects of CSPGs are principally due to the chondroitin sulfate (CS) glycosaminoglycan (GAG) sugar chain (Snow et al., 1990; Cole and McCable, 1991; Geisert and Bidanset, 1993). This is supported by the finding that administration of bacterial chondroitinases in fact promote axon regeneration when administered intrathecally. Moreover, electrophysiological experiments determined that regenerated CST axons established functional connections (Bradbury, et al 2002). In addition to their direct inhibitory effects, CSPGs could also interact with cell adhesion molecules or neurotrophic factors to influence neurite outgrowth (Roberts et al., 1988; Ruoslahti and Yamaguchi, 1991; Milev et al., 1994). Recombinant mammalian chondroitinases is thus useful to reverse the inhibition of CSPG's in the glial scar and to promote axon regeneration following injury.
Bacterial chondroitinases have also been utilized for the treatment of herniated disks in a process known as chemonucleolysis. Chondroitinase ABC can induce the reduction of intradiscal pressure in the lumbar spine. (Sasaki et al., 2001, Ishikawa et al., 1999). There are three types of disk injuries. A protruded disk is one that is intact but bulging. In an extruded disk, the fibrous wrapper has torn and the NP has oozed out, but is still connected to the disk. In a sequestered disk, a fragment of the NP has broken loose from the disk and is free in the spinal canal. Chemonucleolysis is effective on protruded and extruded disks, but not on sequestered disk injuries. In the United States, chemonucleolysis is approved only for use in the lumbar (lower) spine. In other countries, it has also been used successfully to treat cervical (upper spine) hernias. Chemonucleolysis is thus a conservative alternative to disk surgery when it is preferable to reduce disk pressure.
Chondroitinases are enzymes found throughout the animal kingdom. These enzymes degrade chondroitin sulfate through an endoglycosidase reaction. Specific examples of known chondroitinases include chondroitinase ABC (derived from Proteus vulgaris; Japanese Patent Application Laid-open No 6-153947, T. Yamagata, H. Saito, O. Habuchi, and S. Suzuki, J. Biol. Chem., 243, 1523 (1968), S. Suzuki, H. Saito, T. Yamagata, K. Anno, N. Seno, Y. Kawai, and T. Furuhashi, J. Biol. Chem., 243, 1543 (1968)), chondroitinase AC (derived from Flavobacterium heparinum; T. Yamagata, H. Saito, O. Habuchi, and S. Suzuki, J. Biol. Chem., 243, 1523 (1968)), chondroitinase ACII (derived from Arthrobacter aurescens; K. Hiyama, and S. Okada, J. Biol. Chem., 250, 1824 (1975), K. Hiyama and S. Okada, J. Biochem. (Tokyo), 80, 1201 (1976)), chondroitinase ACIII (derived from Flavobacterium sp. Hp102; Hirofumi Miyazono, Hiroshi Kikuchi, Keiichi Yoshida, Kiyoshi Morikawa, and Kiyochika Tokuyasu, Seikagaku, 61, 1023 (1989)), chondroitinase B (derived from Flavobacterium heparinum; Y. M. Michelacci and C. P. Dietrich, Biochem. Biophys. Res. Commun., 56, 973 (1974), Y. M. Michelacci and C. P. Dietrich, Biochem. J., 151, 121 (1975), Kenichi Maeyama, Akira Tawada, Akiko Ueno, and Keiichi Yoshida, Seikagaku, 57, 1189 (1985)), chondroitinase C (derived from Flavobacterium sp. Hp102; Hirofumi Miyazono, Hiroshi Kikuchi, Kelichi Yoshida, Kiyoshi Morikawa, and Kiyochika Tokuyasu, Seikagaku, 61, 1023 (1939)), and the like.
Glycoproteins are composed of a polypeptide chain covalently bound to one or more carbohydrate moieties. There are two broad categories of glycoproteins with carbohydrates coupled through either N-glycosidic or O-glycosidic linkages to their constituent protein. The N- and O-linked glycans are attached to polypeptides through asparagine-N-acetyl-D-glucosamine and serine (threonine)-N-acetyl-D-galactosamine linkages, respectively. Complex N-linked oligosaccharides do not contain terminal mannose residues. They contain only terminal N-acetylglucosamine, galactose, and/or sialic acid residues. Hybrid oligosaccharides contain terminal mannose residues as well as terminal N-acetylglucosamine, galactose, and/or sialic acid residues.
With N-linked glycoproteins, an oligosaccharide precursor is attached to the amino group of asparagine during peptide synthesis in the endoplasmic reticulum. The oligosaccharide moiety is then sequentially processed by a series of specific enzymes that delete and add sugar moieties. The processing occurs in the endoplasmic reticulum and continues with passage through the cis-, medial- and trans-Golgi apparatus.